1. Field of the Invention
This invention relates to a method to prepare biological tissues (e.g. heart valves, veins, cartilage, ligaments) and organs for use as bioprostheses, and more particularly, it relates to a rapid method to preserve tissue samples with a solution, to irradiate samples in a microwave oven, and to store samples in a storage medium.
2. Prior Art
One of the earliest examples of replacement tissue (otherwise known as a bioprosthesis) is the porcine valve which was used to replace defective human heart valves. Before any bioprosthesis is implanted, it must be treated with disinfectants and preservatives (such as chemical, x-ray, or cold treatments) to prevent infection and to maintain the structural integrity of the bioprosthesis. If a bioprosthesis were not treated with preservatives, it would degenerate following implantation and fail to function. In the early stages of this technology, bioprostheses were prepared by immersion in a formaldehyde solution prior to implantation. Using this tanning technology, valves fail to mechanically function properly because the formaldehyde predisposes the valve to either tears in or calcific deposits on the valve leaflets. Levy et al. Am. J. Pathol., 113:143 (1983). The literature teaches that aldehydes are responsible for the mechanical failure of the valve leaflet. The literature currently promotes the use of glutaraldehyde in place of formaldehyde. Furthermore, the literature suggests that reducing the exposure time of the bioprosthesis to the glutaraldehyde or formaldehyde and/or reducing the aldehyde concentration (using conventional tanning methods) would have a limited effect in eliminating mechanical valve failure. Levy et al., Am. J. Pathol., 122:71 (1986).
An effective method of valve sterilization and storage would be one which causes minimal structural change to the collagen and elastic components of the biological tissue. Tan et al., Annals of Thoracic Surg., 22:188 (1976). Collagen is a fibrous protein which gives tissues their structural integrity and makes the tissues resistant to tearing. Collagen fibers form an extracellular framework around tissue cells. Aldehydes induce covalent cross-linking, making collagen fibers rigid as the number of covalent cross-linkages increases.
Aldehydes, as a class, are effective antimicrobial agents, Burke et al., Human Lab Science, 13:267 (1976), which combine a number of organic functional groups on proteins (-NH.sub.2, OH, --COOH, and SH). Spontaneous reactions with the amino groups of proteins leads to protein inactivation by forming methylene-bridged cross-links between amino groups. Sterilization and disinfecting applications of formaldehyde and glutaraldehyde are limited by their slow action (hours) and their uncontrollable reaction rate. Id. In addition, exposure to aldehyde on the order of hours results in irritation of lab workers' respiratory tracts. Science, 222:895 (1983). Prolonged aldehyde immersion is suspected to be responsible for the hardening of collagen, causing tissue to become brittle. Tissue breaks, tears, and calcification are all direct consequences of brittleness. Ferrans et al., Am. J. Cardiology, 41:1159 (1978); Spray et al., Am. J. Cardiology, 40:319 (1977); Tan et al., Ann. Thoracic Surg., 22:188 (1976).
In the field of immunology, low concentrations of glutaraldehyde (0.02% to 0.1%) are used to preserve tissue antigens for immunohistochemical studies. (As used throughout the specification and claims, all percentages are by weight, unless otherwise stated.) Preservation protocols for antigens and enzymes result in poor preservation of the structural integrity of the tissue since these same gentle preservation protocols do not induce a sufficient level of cross-linking. Sabatini et al. J. Cell Biology, 17:19 (1963); Robertson et al., J. Ultrastruct. Research, 30:275 (1970); Wakabayashi et al., J. Histochem. Cytochem., 23:632 (1975).
Preparing tissues by present tanning methods requires long exposure times (hours to months) to moderate glutaraldehyde concentrations (0.625% to 5%). The long exposure time to glutaraldehyde is thought to be necessary for complete penetration of the tissue as well as for tissue sterilization. Mechanical failure, secondary to altered collagen, results in the leading cause of heart valve failure even with glutaraldehyde concentrations as low as 0.625% and with exposure as brief as 24 hours. Broom et al., Thorax, 34:166 (1979).
In the field of histology, the study of tissue structure, the object is to fix cells by preserving their intra- and extracellular architecture. Since the diffusion of glutaraldehyde into tissues is slow (on the order of hours), fixation procedures usually entail long exposure times of tissue specimens to high aldehyde concentrations. This results in very brittle tissue. Glutaraldehyde (in concentrations of 2% to 4%) is used under certain circumstances in histology (e.g. electron microscopy); formaldehyde (in a 4% concentration) is routinely used (e.g. in light microscopy). The goal of fixation is to preserve all cell types and products, not just collagen. That is, histological procedures preserve many cell types and products at the expense of cross-linking collagen, making it shrink and become rigid. Fox et al., J. Histochem. Cytochem., 33:845 (1985). In fact, Ferrans et al. specifically teach that "the concentration of glutaraldehyde used in either the Hancock or in the Carpentier-Edwards process is not sufficiently great to produce tissue fixation of a quality comparable with that needed for ultrastructural study." Ferrans et al., supra.
Recent improvements in the field of histology include the use of microwave energy fixation of surgical specimens for light and electron microscopy. The purpose of the microwave step in fixing specimens is to accelerate the reaction of aldehyde cross-linking with proteins. Boon et al., Histochem J., 20:313 (1988). In this histological procedure, biological specimens are first exposed to solutions of 2-4% formaldehyde and/or glutaraldehyde (typically one hour) prior to microwave heating the solution to temperatures between 50.degree. and 68.degree. C. Leong, Pathology Annual, Part 2, 23:213 (1988); Boon et al., supra. A final microwave fixation temperature of 50.degree. C. or lower is specifically taught against because of the ". . . reduction in brilliance of staining with hematoxylin and eosin." Leong et al., J. Pathol., 146:313 (1985). Then routine tissue processing, paraffin embedding, and staining procedures are performed. Raising the temperature of the specimen and aldehyde using conventional heating sources results in increasing the rate of cell autolysis more so than increasing the rate of diffusion of aldehyde into the specimen. The use of microwave energy in histology is simply a faster method for preparing histological specimens with no other known or reported inherent advantages.
Generally, tissue specimens which are preserved by means of standard tanning processes incorporating glutaraldehyde tend to tear, calcify, and develop thrombi within 4 to 7 years. Riddle et al., J. Thor. Cardiovasc. Surg., 81:279 (1981). Tearing of the bioprosthesis results from repeated flexing of the collagen, made rigid by aldehydes. Calcification is a condition whereby calcium salts from the bloodstream form deposits on stressed or damaged tissues. These deposits develop into hard obstructions within a patient's body, particularly on valve leaflets. A thrombus is a blood clot which is formed at a site of damage to the delicate, internal lining of blood vessels or the heart. Blood clot formation can be triggered by rough surfaces, such as calcified leaflets or tears in the endothelial lining, which cause platelets to release potent clotting factors permitting formation of fibrin fibers which trap red blood cells (erythrocytes) thereby decreasing the function of the leaflets.
The drawbacks to the standard method of using glutaraldehyde to preserve valves are that it induces rigidity in the tissue, making it susceptible to tears in the valves, calcification of the valve leaflets, and induces thrombi following implantation. Leaflets which are less pliable or flexible than natural in vivo valves have poor physiologic function. Current glutaraldehyde preservation techniques require hours to days. Excessive cross-linking and rigidity of collagen in the valve, secondary to prolonged aldehyde exposure time, is considered responsible for the subsequent mechanical failure. Tan et al., supra. The prolonged processing time makes quality control difficult. By storing the valves in a glutaraldehyde solution, an indeterminate amount of additional valve rigidity results as well. Sherman et al., Trans. Am. Soc. Artif. Intern Organs, 30:577 (1984) state that ""control of the extent of cross-linking may represent, therefore, one possible approach to limiting eventual calcification."